Plant Cultivation Method

ABSTRACT

Method for the production of young plants and/or micro-parent stock of 5 herbaceous ornamentals. Method for the production of young plants and/or of micro-parent stock of herbaceous ornamentals, which comprises an in vitro culture phase during which explants obtained from parent stock of species to be propagated, or derivatives of these explants, are subjected to micropropagation which is carried out under suitable conditions and on suitable culture media, in order to produce microplantlets which, when subjected to an in vivo culture phase, are intended to develop into plants or into micro-parent stock, characterized in that, to carry out said micropropagation: the explants are, under axenic conditions, in the dark and for a suitable period of time, placed on a proliferation medium which is composed to suit each plant species to be propagated, so as to induce the formation of white filaments which comprise axillary buds, each of the white filaments is divided into a plurality of pieces, each of 20 which comprises an axillary bud, and said pieces are, in the light, under axenic conditions and for a suitable period of time, placed into a rooting medium, which allows each axillary bud to produce a micro-plantlet which has roots.

The present invention relates to a method for the production of youngplants and/or of micro-parent stock of plants, but especially to plantsbelonging to the group of herbaceous ornamentals, which comprises an invitro culture phase during which explants obtained from parent stock ofplant species to be propagated, or derivatives of these explants, aresubjected to one or more micropropagation cycles which are carried outunder suitable conditions and on suitable culture media, in order toproduce micro-plantlets which, when subjected to an in vivo culturephase, are intended to develop into plants or into micro-parent stock.

It is known that the in vitro micropropagation of a plant in the lightresults in an increased vegetative propagation rate by causing themaximum possible number of axillary buds of a plantlet maintained on aspecific culture medium to break in order to develop shoots.

Methods for the production of plants which are based on an in vitroculture phase in the light and a subsequent in-vivo culture phase havebeen known to the skilled worker for a number of years already. It isrecognized in the art that these methods have a number of advantageswhich is basically a result of going through an in vitro phase and whichis reflected not only in terms of the quantities of plants produced perarea, but also in terms of plant health, quality and final appearance ofthe plant. However, their use in horticulture is currently still greatlyrestricted since each type of plant grown requires specific cultureconditions the development of which involves a great deal of time andeffort and is therefore costly. The propagation rates that can beachieved with said methods are not yet sufficient to compensate forthese disadvantages.

Moreover, it has been found that certain plant species such as, inparticular, pelargonium, for which the production via this route hasbeen attempted do not lend themselves readily to known micropropagationmethods.

One of the objectives of the present invention is, therefore, to proposea process for the production of young plants and/or of micro-parentstock of plants which process is perfectly adapted to the culture ofsaid plants, but especially of plants belonging to the group ofherbaceous ornamentals such as, in particular, pelargonium, petuniaincluding cascading petunia, poinsettia, cyclamen, chrysanthemum, busyLizzie, verbena, or torenia, such as to achieve increased propagationrates.

Moreover, it is another objective of the invention to provide a methodthe application of which would make it possible to achieve proliferationrates, that are much higher than those obtained so far with methodswherein the proliferation phase is performed in the light.

Moreover, it is also an objective of the invention to propose a methodfor the production of young plants and/or of micro-parent stock ofplants, but especially of plants belonging to the group of herbaceousornamentals, which method integrates a particular in vitromicropropagation technique with the aid of which more vigorous youngplants or parent stock with an exceptionally high branching rate andmore rapid growth can be obtained.

Another object of the invention is to propose a vegetative propagationmethod which is suitable for ensuring the health status and themaintenance of genetic criteria, but especially genetic conformity ofthe plants over the generations, resulting in phenotypically uniform andproperly synchronized plant populations.

The in vitro culture phase according to the present invention is alsosuitable for mechanization owing to the miniaturization of the cuttingproduction that can be achieved by applying the method according to thepresent invention. Plants can be supplied in high density trays, whichcan be suitably used in automated transplanting systems involvingtransplanting robots.

Finally, the method, which is the subject of the present invention isalso directed at improving the financial returns of the production ofplants by reducing the time span and area required for their propagationby carrying out an in vitro micropropagation under specific conditions.

In one embodiment, the invention relates to a method for the productionof young plants and/or of micro-parent stock of plants, but especiallyplants belonging to the group of herbaceous ornamentals, which methodcomprises an in vitro culture phase during which explants obtained fromparent stock of species to be propagated, or derivatives of theseexplants, are subjected to a micropropagation phase, which is carriedout under suitable conditions and on suitable culture media, in order toproduce micro-plantlets which, when subjected to an in vivo culturephase, are intended to develop into plants or into micro-parent stock,characterized in that, to carry out said micropropagation:

(a) explants are obtained from parent stock of species to be propagated,or from derivatives of these explants,(b) the explants are, under axenic conditions, placed on a shootinitiation medium which is composed such as to suit each plant speciesto be propagated, and, in the dark and for a suitable period of time,grown so that the formation of white filaments is induced which compriseaxillary buds,(c) optionally, each of the white filaments is then divided into aplurality of segments or pieces, each of which comprises an axillarybud,(d) and the white filaments and/or pieces or segments thereof are, inthe light, under axenic conditions and for a suitable period of time,placed into or on a rooting medium, which allows each axillary bud toproduce a micro-plantlet which has roots.

In another embodiment, the method according to the invention, involvinga micropropagation step, is characterized in that

(a) explants are obtained from parent stock of species to be propagated,or from derivatives of these explants,(b) the explants are, under axenic conditions, placed on a shootinitiation medium which is composed such as to suit each plant speciesto be propagated, and, in the dark and for a suitable period of time,grown so that the formation of white filaments is induced which compriseone or more axillary buds,(c) optionally, each of the white filaments is then divided into aplurality of segments or pieces, each of which comprises an axillarybud, but at least one axillary bud,(d) the white filaments and/or pieces or segments thereof obtained instep b) are, under axenic conditions transferred to a proliferationmedium and grown in the dark for a suitable period of time sufficient toallow the tissue of the plant cuttings to multiply, preferablyexponentially, and to produce a large quantity of etiolated whitefilaments comprising a high number of axillary nodes, which give rise toplantlets intended to develop during an “in vivo” phase if grown inlight; or to develop into shoots with the axillary shoots becomingetiolated into white filaments, if grown in the dark,(e) this step (d) is repeated as often as necessary to produce differentconsecutive generations of white filaments until the desired amount ofwhite filaments has been obtained,(f) and the white filaments or pieces thereof are, in the light, underaxenic conditions and for a suitable period of time, placed into or on arooting medium, which allows each axillary bud to produce amicro-plantlet which has roots.

The initiation medium used above in step (b) and the proliferationmedium mentioned in step (d) may have an identical or an essentiallyidentical composition or may be different media, depending on therequirements of the plant species or genotype used in themicropropagation method according to the invention.

Conditions and culture media that can be suitably used in plantmicropropagation are well known to those skilled in the art of plantcultivation and are described, for example, in “Plant Propagation byTissue Culture, Handbook and Directory of Commercial Laboratories, eds.Edwin F George and Paul D Sherrington, Exegetics Ltd, 1984”.

In a further aspect of the present method, a traditional culture medium,but especially a culture medium of the “MS” type, either supplementedonly with a compound which belongs to the cytokinin family of growthregulators or supplemented with a compound which belongs to thecytokinin family of growth regulators and with a compound which belongsto the auxin family of growth regulators is used as an initiation and/ora proliferation medium in the micropropagation phase. Said growthregulating compounds are specifically chosen and provided in the mediumin a concentration, which promotes proliferation of the plant materialfrom the plant species to be multiplied, but especially the developmentof white filaments from the axillary buds or nodes initially present onthe explant material and elongation growth of said white filaments suchthat a maximum number of axillary buds/per initial bud or node areproduced on the developing filaments, but at least between 2 and 10axillary buds/per initial bud, specifically between 3 and 7 axillarybuds/per initial bud, but especially between 4 and 5 axillary buds/perinitial bud, sustaining a high multiplication rate.

Compounds which belong to the cytokinin family of growth regulators andwhich may be used in the method according to the present invention as asuitable supplement for the initiation and/or proliferation medium maybe any compound selected from the group consisting of purin-basedcytokinins such as, for example, kinetin, zeatin, 6-benzylamino purine(BAP); 6-(benzylamino)-9-(2-tetrahydropyrany10-9h-purine (PBA); or(6-(γ,γ-dimethylallylamino) purine (2iP); and cytokinins based onsubstituted phenyl ureas such as, for example, thidiazuron (TDZ) or anyother compound belonging to the cytokinin family of growth regulatorswhich is know or demonstrated to be suitable for directly or indirectlypromoting the propagation potential of the plant species to bemultiplied, but especially the development of white filaments from theaxillary buds or nodes initially present on the explant material andelongation growth of the developing white filaments such that a maximumnumber of axillary buds/per initial bud or node are produced on thedeveloping filaments, but at least between 2 and 10 axillary buds/perinitial bud, specifically between 3 and 7 axillary buds/per initial bud,but especially between 4 and 5 axillary buds/per initial bud, sustaininga high multiplication rate.

Compounds which belong to the auxin family of growth regulators andwhich may be used in the method according to the present invention as asuitable supplement for the initiation and/or proliferation medium maybe any compound selected from the group consisting of indole-3-butyricacid (IBA); α-naphthaleneacetic acid (NAA); indole-3-acetic-acid (IAA);and 2,4-dichlorophenoxyacetic acid (2,4-D) or any other compoundbelonging to the auxin family of growth regulators which is know ordemonstrated to be suitable for directly or indirectly promoting thepropagation potential of the plant species to be multiplied, butespecially the development of white filaments from the axillary buds ornodes initially present on the explant material and elongation growth ofsaid white filaments such that a maximum number of axillary buds/perinitial bud or node are produced on the developing filaments, but atleast between 2 and 10 axillary buds/per initial bud, specificallybetween 3 and 7 axillary buds/per initial bud, but especially between 4and 5 axillary buds/per initial bud, sustaining a high multiplicationrate.

Further compounds that may be suitably used as a supplement for theinitiation and/or proliferation medium are, for example, inositol, butespecially one or more of its nine distinct isomers commonly found inplant and/or animal systems, such as myo-inositol, further, biotine,folic acid, cystein or polyvinyl pyrrolidone (PVP) or any other compoundwhich is know or demonstrated to directly or indirectly support growthand/or the propagation of the plant species to be multiplied.

Another compound that may be used as a supplement in the propagationmedium according to the invention is a suitable carbon source,especially a sugar compound such as, for example, sucrose or glucose orany other sugar compound commonly used in plant cultivation or acombination thereof.

Yet, the present method is further characterized by the fact that whitefilaments are obtained comprising axillary buds and that said firstgeneration of white filaments, but especially segments or pieces of saidfilaments obtainable through, for example, cutting of the filaments intoa plurality of defined pieces or segments containing at least oneaxillary bud, are returned under axenic conditions and in the dark to afresh proliferation medium and cultivated in the dark for a suitableperiod of time so as to induce the formation of a new generation ofwhite filaments such that a maximum number of axillary buds/per initialbud present on the first generation filament are produced on thedeveloping filaments, but at least between 2 and 10 axillary buds/perinitial bud, specifically between 3 and 7 axillary buds/per initial bud,but especially between 4 and 5 axillary buds/per initial bud, sustaininga high multiplication rate. The procedure may be repeated as often asnecessary to produce different consecutive generations of whitefilaments until the desired amount of white filaments has been obtained.

Thus, the present invention makes it advantageously possible to proposea technique which can be defined for a given plant species, inparticular a plant species belonging to the group of herbaceousornamentals by a sequence of standardized operations in which the typeof the media and the culture conditions may, if required, bespecifically adapted to suit varieties or groups of varieties withinsaid species in order to increase the vegetative propagation rate andmaintain this high propagation rate over one or more, specifically overup to 10, more specifically over up to 5, propagation cycles in thedark.

Plants that can be used in a process according to the present inventionare plants that belong to the group of herbaceous ornamentals such asplants of the families Adiantaceae Amaranthaceae, Amaryllidaceae,Apiaceae, Apocynaceae, Araceae, Araliaceae, Asclepiadaceae,Aspleniaceae, Asteraceae, Athyriaceae, Balsaminaceae, Begoniaceae,Buxaceae; Campanulaceae, Cannaceae, Carophyllaceae, Crassulaceae,Dryopteridaceae, Euphorbiaceae, Fumariaceae, Geraniaceae, Hyperaceae,Iridaceae, Lamiaceae, Liliaceae, Lobeliaceae; Nyctaginaceae,Osmundaceae, Piperaceae, Plumbaginaceae, Poaceae, Portulacaceae,Ranunculaceae, Rosaceae, Saururaceae, Saxifragaceae, Scrophulariaceae,Solanaceae, Verbenaceae, and Violaceae, in particular, plants belongingto the group of pelargonium, petunia including cascading petunia,poinsettia, cyclamen, chrysanthemum, busy Lizzie, verbena, or torenia,but especially pelargonium.

In one embodiment of the present invention, implementation of thepresent method results in a first variant wherein, in order to obtainbacteria- and virus-free parent stock, meristems, specifically growingmeristems, but especially shoot meristems, are taken from parent stockand used as explants which are grown under axenic conditions in thelight for a first period of time on a development medium common to eachplant variety, sufficient to bring about the formation of littleplantlets which have first leaf primordia and then, for a second periodof time, on or in a rooting medium common to each plant variety, inorder to obtain rooted plantlets which are free from bacteria andviruses and which are intended to be placed on a shoot initiation mediumin the dark in order to produce said white filaments according to theinvention.

Conditions and culture media that can be suitably used in plant meristemculture as well as for rooting plantlets obtained by said culture arewell known to those skilled in the art of plant cultivation and aredescribed, for example, in “Plant Propagation by Tissue Culture,Handbook and Directory of Commercial Laboratories, eds. Edwin F Georgeand Paul D Sherrington, Exegetics Ltd, 1984”.

In this situation, according to a characteristic of the present method,the volume of the rooting medium used is supplemented in a suitablemanner during the rooting phase of said plantlets. Rooted plantlets arethen transferred to soil or any other suitable supporting mediumcommonly employed in plant cultivation practice for growing youngplants, acclimatized to changed environmental conditions such as thosepresent in a greenhouse and grown there for a suitable time period, butat least for 4 weeks to 10 weeks, especially for 5 weeks to 6 weeks.

The plants may then be subjected to a suitable testing system in orderto detect and discard plant material that is infected with viral and/orbacterial pathogens such as, for example, an “ELISA”-type test and anysamples which test positive are eliminated, while those which testnegative are retained as bacteria- and virus-free parent stock and usedas a source of explants.

In one embodiment of the invention, the leaves and roots of saidplantlets are suppressed for a suitable period of time before beingplaced, under axenic conditions and in the dark, on a shoot initiationmedium which is specific to each variety in order to produce said whitefilaments.

In another embodiment of the invention, micro-cuttings are taken fromthe bacteria- and virus-free parent stock plants obtainable through ameristem culture as described hereinbefore and used as explants.Alternatively, certified bacteria- and virus-free parent stock that maybe purchased from a commercial source may be used for obtainingmicrocuttings. In particular, branches, but especially shoot tipscomprising one or more axillary buds are taken and cut into pieces witheach piece containing at least one bud.

In accordance with a specific embodiment of the invention, the leavesand the stipules of the apical portion of the micro-cuttings aresuppressed for a suitable period of time before being placed, underaxenic conditions and in the dark, on a shoot initiation medium which isspecific for each plant variety, in order to produce said whitefilaments.

In accordance with another advantageous characteristic of the presentmethod, the white filaments which have axillary buds may be stored for agiven period of time before the rooting phase during which each budproduces a rooted micro-parent stock plant which is intended for beingcultured in vivo.

Secondly, the present invention also relates to the micro-plantlets,young plants and micro-parent stock obtained by carrying out the methodas defined hereinabove.

In a specific embodiment, the present invention relates to young plantswhich are distinguished in particular by an increased branching rate,rapid growth, compact foliage, auto-regulation of the plant, butespecially to young plants selected from the group consisting ofpelargonium, petunia including cascading petunia, poinsettia, cyclamen,chrysanthemum, busy Lizzie, verbena, and torenia.

The present invention also relates to the characteristics which will beseen from the description to follow, and which must be considered inisolation or in accordance with any possible combinations thereof.

The method according to the invention comprises an “in vitro” culturephase during which plant tissues are, under axenic conditions, subjectedto different treatments, in particular of micropropagation, whichtreatments are carried out under specific conditions to make these planttissues multiply exponentially and produce a large quantity of plantletswhich are intended to develop during an “in vivo” phase, either directlyinto plants or into micro-parent stock intended to act itself asstarting material for obtaining plants.

The method according to the invention is further expected to be suitablefor being applied either to plantlets obtained from meristems taken fromany parent stock, or to micro-cuttings taken from healthy parent stock,viz. certified, bacteria- or virus-free parent stock.

Thus, the variant of the method which relies on the use of meristems andwhich has the advantage of leading to a sanitization of parent stockwhich may be attacked by any pathogen requires supplementary steps ofobtaining plantlets. To this end, meristems, but especially developedmeristems with the first leaf primordia are obtained and first grown fora period of approximately 4 months in the light in a suitable containersuch as, for example, a Petri dish, glass jar or pot, on a developmentmedium whose composition is advantageously common to all the plantspecies to which the present method relates, for example, one of thetraditionally used plant cultivation media such as, for example, a“MS”-type culture medium (MS macro- and micro-elements; MS vitamins)which is supplemented with compounds which belong to the cytokininfamily of growth regulators.

Small plantlets obtained from the developed meristems, are subsequentlyplaced on or in a rooting medium whose composition is identical,whatever the plant species, preferably a rooting medium traditionallyused in plant cultivation such as, for example, a culture medium of the“MS”-type, in the light, to obtain small plantlets, which, approximatelyafter one month, are transferred to a suitable container such as, forexample, a glass jar or glass pot, which allows the root volume toincrease. To further promote root development ventilated container maybe used.

After this procedure, rooted plantlets are obtained which are ready toenter the actual in vitro micropropagation phase, in the same way as themicro-cuttings taken from healthy parent stock such as, for example,certified mother stock plants grown in the greenhouse under controlledphytosanitary conditions.

In a specific embodiment of the invention, the plantlets ormicro-cuttings are, before being subjected to a micropropagationtreatment, first rejuvenated by, for example, suppressing the roots andthe leaves and then placed on a shoot initiation medium which isdeveloped specifically to suit the plant variety to be propagated andwhich is spread out in a suitable container such as a Petri dish.

In particular, leafs and, if present, roots are removed from the stem orbranches of the plant to be micropropagated and, preferably, surfacesterilized. They are then cut into pieces or segments with each piece orsegment containing at least one node or axillary bud. The cuttings aretransferred on to a shoot initiation medium and cultivated in the darkunder conditions and for a period of time, which allow the plantcuttings to multiply and produce a large quantity of etiolated whilefilaments comprising a high number of axillary nodes, which give rise toplantlets intended to develop during an “in vivo” phase if grown inlight; or to develop shoots with the axillary shoots becoming etiolatedinto white filaments, if grown in the dark.

The cultivation in the dark of plant cuttings as well as cuttingsobtained from etiolated white filaments comprising at least one axillarybud or node is carried out for a period of time sufficient to allow forthe axillary nodes or axillary buds to develop into shoots and theaxillary shoots to become etiolated into white filaments comprisingaxillary buds, with the number of axillary buds produced on thedeveloping filaments amounting to at least between 2 and 10 axillarybuds/per initial node present in the cutting, specifically between 3 and7 axillary buds/per initial node present in the cutting, but especiallybetween 4 and 5 axillary buds/per initial node present in the cutting,but at least for a period of between approximately 2 weeks toapproximately 12 weeks, specifically of between approximately 3 weeks toapproximately 7 weeks, more specifically of between approximately 4weeks to approximately 5 weeks, but especially of approximately 4 weeks.

In a specific embodiment, for shoot initiation and maintenance the plantexplants are placed horizontally on the cultivation medium and grownunder conditions, which forces the developing shoots to grow essentiallyhorizontally. This can be achieved by growing the explants in acontainer such as a Petri dish, which is designed such as to prevent theexplant from growing vertically owing to space limitations in thevertical direction. For the developing shoots it is preferable to be inclose contact with the cultivation medium and to establish more thanjust one contact point with the medium during micropropagation.

In accordance with the present method, it is advantageous to use aninitiation and/or a proliferation medium which has substantially thesame basic composition as the above-mentioned development medium androoting medium, namely a composition based on the traditional presenceof macro- and micro-elements and vitamins such as, for example, thosepresent in the commonly used MS media.

The composition and concentration of the macro- and micro-elements andvitamins within said media may vary to a certain extent depending on theplant species or genotype involved. Such minor adaptations are wellwithin the skills of the skilled artisan in the area of plantcultivation and part of his routine optimization work. For example, whenusing an MS-type medium it has proven advantageous in some instances toemploy reduced concentrations of macro- and/or micro-elements and/orvitamins, especially a concentration which establishes itself somewherebetween a full-strength and a half-strength concentration, but inparticular, a half-strength concentration.

Said basic composition may be further supplemented by the addition ofcompounds belonging to the cytokinin family of growth regulators or bythe simultaneous addition of compounds belonging to the cytokinin familyof growth regulators and compounds belonging to the auxin family ofgrowth regulators, depending on the plant species to be propagated,which compounds support shoot multiplication by promoting the breakageand development of axillary buds into etiolated shoots and further intowhite filaments and/or the growth, but especially the elongation growthof the developing shoots into shoots comprising a high number ofaxillary nodes.

In a specific embodiment, a two-layer cultivation approach is employedin the method according to the invention wherein the plant cultivationmedium is provided in form of a two layer system comprising a solidifiedbottom layer comprising essentially macro- and micro-elements, vitaminsand a carbon source as described herein below, but no growth regulator,which is overlayed with a fluid top layer comprising a growth regulator,especially a growth regulator belonging to the cytokinin family ofgrowth regulators or a combination of growth regulators belonging to thecytokinin family of growth regulators and to the auxin family of growthregulators, depending on the plant species to be propagated, whichgrowth regulators support shoot multiplication by promoting the breakageand development of axillary buds into etiolated shoots (white filaments)and/or the growth, but especially the elongation growth of thedeveloping shoots into shoots comprising a high number of axillarynodes.

The fluid top layer may, in addition to the growth regulators belongingto the cytokinin and/or the auxin family of growth regulators of growthregulators, contain other growth regulators of a different family ofgrowth regulators such as, for example, growth regulators belonging tothe gibberellin family of growth regulators, which are known to promoteshoot elongation.

The fluid top layer may further contain compounds supporting growth ofthe developing shoot such as, for example, macro- and micro-elements,vitamins and a carbon source. The volume of the fluid top layer ascompared to the solid bottom layer amounts to between about 1% and about10%, specifically between about 2% and about 7%, more specificallybetween about 3% and about 5%, but especially between about 3% and about4%.

In a specific embodiment of the invention, the two layer cultivation iscarried out in a suitable container commonly used in plant cultivationsuch as, for example, a glass jar or glass pot with a size big enough toallow the developing white filaments to grow properly. The containersmay, in addition, be ventilated in order to support growth anddevelopment of the white filaments.

Compounds which belong to the cytokinin family of growth regulators andwhich may be used in the method according to the present invention as asuitable supplement for the initiation and/or proliferation medium maybe any compound selected from the group consisting of kinetin, zeatin,6-benzylamino purine (BAP);6-(benzylamino)-9-(2-tetrahydropyrany10-9h-purine (PBA);(6-(γ,γ-dimethylallylamino) purine (2iP); and thidiazuron (TDZ) or anyother compound belonging to the cytokinin family of growth regulatorswhich is know or demonstrated to be suitable for propagation of theplant species to be multiplied.

Compounds belonging to the cytokinin family of growth regulators areoffered in the initiation and/or proliferation medium according to theinvention in a concentration of between 0.01 mg/l and 5.0 mg/l-7.0 mg/l,specifically between 0.03 and 2.0 mg/l, more specifically between 0.04and 1.0 mg/l, but especially between 0.05 and 0.5 mg/l.

Adaptations may need to be made within the above defined ranges in orderto accommodate the specific needs of the plant species to be multiplied.

Compounds which belong to the auxin family of growth regulators andwhich may be used in the method according to the present invention as asuitable supplement for the initiation and/or proliferation medium maybe any compound selected from the group consisting of indole-3-butyricacid (IBA); a-naphthaleneacetic acid (NAA); indole-3-acetic-acid (IAA);and 2,4-dichlorophenoxyacetic acid (2,4-D) or any other compoundbelonging to the auxin family of growth regulators which is know ordemonstrated to be suitable for propagation of the plant species to bemultiplied.

Compounds belonging to the auxin family of growth regulators are offeredin the initiation and/or proliferation medium according to the inventionin a concentration of between 0.01 mg/l and 5.0 mg/l, specificallybetween 0.03 and 2.0 mg/l, more specifically between 0.04 and 1.0 mg/l,but especially between 0.05 and 0.5 mg/l.

Adaptations may need to be made within the above defined ranges in orderto accommodate the specific needs of the plant species to be multiplied.

Some of the growth promoting substances such as compounds belonging tothe group of the substituted phenyl ureas, for example thidiazuron(TDZ), alone or in combination with another compound of the cytokininfamily of growth regulators such as, for example, 6-benzylamino purine(BAP) are known to be potent stimulators of multiple shoot formation.These substances are further known to be rather persistent and highlyactive already at low concentrations in a range of between 0.001 mg/land 0.1 mg/l but may, in some cases, cause some undesirable effectslike, for example, organ aberrations or stunted shoot growth.

These undesired effects may be compensated for by the addition of agibberellin-type growth regulator such as gibberellic acid, whichpromotes shoot elongation.

Another possibility to avoid undesired effects caused by the presence ofgrowth regulators, but especially growth regulators which may negativelyaffect shoot growth and elongation such as, for example, TDZ, in theinitiation and/or the proliferation medium, is the application of atwo-layer cultivation approach as mentioned hereinbefore, whereby thecompounds belonging to the group of the substituted phenyl ureas suchas, for example, thidiazuron (TDZ), alone or in combination with anothercompound of the cytokinin family of growth regulators such as, forexample, 6-benzylamino purine (BAP) are only present in the fluid toplayer in a concentration high enough to promote shoot multiplication butlow enough to prevent any undesired effects to occur, but specificallyin a concentration of approximately between 0.001 mg/l and 2.0 mg/l,more specifically between approximately 0.005 and 1.0 mg/l, butespecially between 0.01 and 0.1 mg/ml.

Further compounds that may be suitably used as a supplement for theinitiation and/or proliferation medium are, for example, inositol, butat least one of its nine distinct isomers such as myo-inositol, further,biotine, folic acid, cystein or polyvinyl pyrrolidone (PVP) or any othercompound which is know or demonstrated to directly or indirectly promotegrowth and/or the propagation potential of the plant species to bemultiplied.

Another compound that may be used as a supplement in the propagationmedium according to the invention is a suitable carbon source,especially a sugar compound such as, for example, sucrose or glucose, orany other sugar compound commonly used in plant cultivation, or acombination thereof.

The white filaments obtained in this or any further propagation cyclescan be placed in the dark under conditions that allow preservation ofthe filaments for later rooting or as stock for further propagationrounds. For example, filaments stored at a temperature of between 2° C.and 10° C., specifically of between 3° C. and 8° C., but especially ofbetween 4° C. and 6° C. may be stored for at least up to 10 month,specifically for at least up to 7 month, but especially for at least upto 5 months.

Thus, the micropropagation phase can advantageously be carried outagain, as often as necessary in cycles of between approximately 3 weeksto 12 weeks, specifically of between approximately 4 weeks to 6 weeks,but especially of between approximately 4 weeks to 5 weeks, in order toobtain large quantities of white filaments.

In accordance with a further characteristic of the present method, whichallows the quality of the plants produced to be ensured in terms ofhealth, plants resulting from the cultivation of meristems and/or thewhite filaments are subjected to a suitable testing system such as, forexample, an “ELISA”-type test and any samples which test positive areeliminated, while those which test negative are retained.

Secondly, in accordance with another advantageous characteristic, it ispossible to store for as long as necessary the portions of whitefilaments in the dark at ambient temperature on a proliferation mediumwhich is replaced periodically, before the rooting phase during whicheach bud produces a rooted micro-plantlet which is intended forculturing in vivo.

Several plant species have already been subjected to a micropropagationtreatment in accordance with the method of the present invention.

Thus, it has been possible to demonstrate that, by using a medium of thetraditional “MS”-type supplemented with cytokinins or a combination ofcytokinins and auxins as a shoot initiation and a proliferation medium,micro-plantlets of pelargonium, for example, of zonal geranium, ofdouble ivy-leaved geranium, or of simple ivy-leaved geranium, as well aspetunia and osteospermum micro-plantlets have successfully beenproduced.

Moreover, the method according to the invention can also be applied toother plant species belonging to the group of herbaceous ornamentalssuch as, for example, plants of petunia including cascading petunia,kalanchoe, torenia, verbena and rose-bush to successfully producemicro-plantlets.

Moreover, by applying the method according to the invention it has alsobeen found that the young plants or micro-parent stock obtained frommicro-plantlets had particularly interesting characteristics.

The plants produced through this method naturally present a very highdegree of branching, rapid rooting, homogeneous growth in terms ofvolume, rapid hardening-off of the plant, a leaf area which is smallerthan that of plants obtained in traditional culture methods, and apronounced juvenile character, which can be seen in particular frommicro-plantlets of zonal geranium, a micro-parent stock of zonalgeranium and a micro-parent stock plant of cascading petunia,respectively.

The present method also offers an advantageous solution which makespossible an improved productivity and flexibility in the productionprocess as compared with the traditional culture techniques, inparticular by reducing the surfaces which are traditionally allocated tothe culture of parent stock by a factor of four.

Moreover, by applying the method according to the invention it would bepossible to achieve proliferation rates, that are much higher than thoseobtained so far performing the proliferation phase in the light. If oneassumes that each white filament obtained from a starting plantlet isable to provide at least five axillary buds per month, which,themselves, are capable of providing five rooted micro-plantlets, eachof which, in turn, is capable of providing five white filaments permonth, this would lead, after 9 micropropagation cycles of one month, toapproximately 2 million plants obtained from a single starting plantlet.

Moreover, the implementation of the present method further offers manyadvantages which are reflected in the different horticultural networksconcerned, in particular, in an improvement of the administration oforders, and in the higher plant quality.

Although the invention has been described with reference to a particularembodiment, it is well understood that it is in no way limited theretoand that various modifications of form, materials and combinations ofthese various elements can be made thereto without, however, departingfrom the scope or the spirit of the invention.

The following working examples refer to examples of implementing thepresent method, which are given by way of illustration and not by way oflimitation and which relate to the production of pelargonium, petuniaand osteospemum plants, more specifically to the production of plantswhich belong to different varieties or genotypes of pelargonium, petuniaand osteospemum.

EXAMPLES Example 1 Petunia 1.1 Plant Material and Etiolation

Petunia certified motherstock plants are grown in the greenhouse for atleast 6 weeks under controlled phytosanitary conditions. Branches areobtained from said motherstock plants and used as explants for shootmultiplication and etiolation.

All the leaves are removed from the branches and the defoliated branchesare surface sterilized with a 1.3% sodium hypochlorite solution (30%commercial bleach) for 10 minutes and washed three times with steriledistilled water.

After sterilization, the defoliated branches are cut into pieces underaxenic conditions with each piece containing one or two nodes.

Explants are placed horizontally on a solidified shoot initiation mediumin a petridish, with a maximum of three explants per petridish.Petridishes are sealed with nescofilm.

For etiolation two MS-based media are used for shoot initiation, MS1 andMS1-2. The two media essentially differ in the concentration of the MSmacro elements in that the MS1-2 medium contains the macro elements onlyin half-strength concentration. Both media can be used for shootinitiation and etiolation of Petunia explants, even though certaingenotypes appear to have preferences for one or the other medium.

The MS1 medium contains MS micro salts+MS macro salts+MS vitamins,sucrose as a carbon source, myo-inositol, biotin, and folic acid asadditional, non-MS vitamins, PVP (polyvinyl pyrrolidone) as a furthersupplement and BAP (6-benzylaminopurine) and 2iP (2-isopentenyl-adenine)as growth hormones of the cytokinin family of growth regulators. Themedium is solidified with plant agar. The pH is set at 5.8 beforeautoclaving (for further details of the medium composition see table Abelow).

The M1-2 medium contains half strength MS macro salts+full MS microsalts and MS vitamins, sucrose as a carbon source, myo-inositol, biotin,and folic acid as additional, non-MS vitamins, PVP (polyvinylpyrrolidone) as a further supplement and BA (6-benzylaminopurine) and2iP (2-isopentenyl-adenine) as growth hormones of the cytokinin familyof growth regulators. The medium is solidified with plant agar. The pHis set at 5.8 before autoclaving (for further details of the mediumcomposition see table A below).

Explants are grown in the dark for 4 weeks at a temperature of between18° C. and 20° C. In general 60%-90% of the explants will form filaments(see table 1 below).

After four to five weeks cultivation in the dark the axillary shoots areetiolated into white filaments. Each filament has one or more axillarynodes.

TABLE 1 Number of filaments per explant after 4 weeks genotype MS1 MS1-2A971-1 3.0 5.0 A895-3 4.5 8.0 A1019-1 — 6.2 A938-5 5.0 1.0 A1010-3 5.017.0 A948-1 6.0 10 average 3.9 7.9

1.2 Proliferation

For maintenance, the white filaments obtained in Example 1.1 are cutinto pieces containing one or two axillary nodes and transferred tofresh MS1 or MS1-2 medium in petridishes. For preserving the stock thepetridishes can be placed in the dark at 4° C. for at least 4 months.

For an efficient proliferation filaments are used which contain morethan one axillary node. However, satisfactory results could also beobtained with filaments containing only one axillary node as thestarting material.

The filaments or pieces thereof obtained through cutting of thefilaments comprising between 2 and 6 axillary nodes are grown in glassjars containing 66 ml medium. The medium is SP1, which contains modifiedMS macro salts, MS micro salts+MS vitamins, sucrose as the carbonsource, myo-inositol, biotin, folic acid as additional non-MS vitamins,BAP (6-benzylaminopurine) as a representative of the cytokinin family ofgrowth regulators, and IAA (Indole-3-Acetic Acid) as a representative ofthe auxin family of growth regulators. The medium is solidified withplant agar (for further details of the medium composition see table Abelow).

6 filaments or pieces thereof are placed in one jar, with the first nodejust below the surface of the agar. The jars are placed in the dark at atemperature of between 18° C. and 22° C. for 5-6 weeks. After five toeight weeks further subcultivation cycles can be carried out byfollowing essentially the same procedure as described above.

At each subculture the proliferation rate or multiplication rate can bedetermined by dividing the number of filaments formed (after separatingor cutting the tissue back to the original number of nodes) by theinitial number of filaments. The proliferation rates are determined for6 genotypes for 2 consecutive subcultures of between 4 to 5 weeks (seetable 2 below).

TABLE 2 Proliferation rates of genotypes subculture 1 2 A971-1 7.0 5.5A895-3 3.0 4.8 A1019-1 3.0 4.2 A938-5 1.0 5.8 A1010-3 2.0 1.4 A948-1 3.58.0 average 3.3 5.0

1.3 Rooting

For rooting smaller explants are used. Each filament contains one or twoaxillary nodes. The explants are transferred to ventilated containers(ecoline boxes with XXL filter). The rooting medium is MS0-2 (halfstrength MS macro salts, full strength MS micro salts and MS vitamins,sucrose as the carbon source, myo-inositol, biotin, folic acid asadditional non-MS vitamins, and plant agar as the solidifying agent) orRAI2-2 (half strength MS macro salts, full strength MS micro salts andMS vitamins, sucrose as the carbon source, myo-inositol, biotin, folicacid as additional non-MS vitamins, indole-acetic acid, indole-butyricacid as growth regulators of the auxin family of growth regulators, andplant agar as the solidifying agent).

Both media can be used and will give similar results. Plants are rootedfor four weeks at a temperature of between 18° C. and 22° C. in a 16 hphotoperiod of app. 3000 lux. Rooting percentages are determined after4-6 weeks.

The rooted plantlets are taken out of the containers and washed in waterto remove traces of medium and agar. The plantlets are transferred tosoil in the greenhouse at 20° C. where they acclimatized and are treatedas normal tissue culture plants.

Example 2 Osteospermum 2.1 Plant Material and Etiolation

Osteospermum certified motherstock plants are grown in the greenhousefor at least 6 weeks under controlled phytosanitary conditions. Branchesare obtained from said motherstock plants and used as explants for shootmultiplication and etiolation.

All the leaves are removed from the branches and the defoliated branchesare surface sterilized with a 1.3% sodium hypochlorite solution (30%commercial bleach) for 10 minutes and washed three times with steriledistilled water.

After sterilization, the defoliated branches are cut into pieces underaxenic conditions, with each piece containing one or two axillary nodes.Explants are placed horizontally on a solidified shoot initiation mediumin a petridish, with a maximum of two explants per petridish.Petridishes are sealed with nescofilm.

For etiolation two MS-based media are used, MS1-2 and MS2Z, whichessentially differed in the concentration of the MS macro elements andthe growth hormone composition. Both media can be used for etiolation ofOsteospermum explants, even though certain genotypes appear to havepreferences for one or the other medium.

The M1-2 medium contains half strength MS macro salts+full strength MSmicro salts and MS vitamins, sucrose as a carbon source, myo-inositol,biotin, and folic acid as additional vitamins, PVP (polyvinylpyrrolidone) as a further supplement, and BAP (6-benzylaminopurine), and2iP (2-isopentenyl-adenine) as representatives of growth factors of thecytokinin family of growth regulators. The medium is solidified byaddition of plant agar. The pH is set at 5.8 before autoclaving (forfurther details of the medium composition see table A below).

The MS2Z medium contains full strength MS micro+MS macro salts+MSvitamins, sucrose as a carbon source, myo-inositol, biotin, and folicacid as additional non-MS vitamins, and zeatine as the growth factor ofthe cytokine family of growth regulators. The medium is solidified byaddition of plant agar. The pH is set at 5.8 before autoclaving (forfurther details of the medium composition see table A below).

Explants are grown in the dark at a temperature of 18° C. (see table 3below). After four weeks of cultivation in the dark the axillary shootsare etiolated into white filaments. Each filament has one or moreaxillary nodes.

TABLE 3 Number of filaments per explant after 4 weeks genotype MS1-2MS2Z E192-1 0.75 0 D209-3 0 0 E207-2 3.3 2.2 E206-1 0.6 0.3 average 1.20.6

2.2 Proliferation 2.2.1 Single Layer Cultivation

The white filaments obtained in Example 2.1 are cut into piecescontaining one or two nodes and transferred to fresh MS1-2 inpetridishes for maintenance after four-five weeks. For preserving thestock the petridishes can be placed in the dark at 4° C. for at least 4months.

For an efficient proliferation filaments are used which contain at leasttwo axillary nodes. The further steps are essentially carried out asdescribed in Example 1.2 for Petunia.

TABLE 4 Proliferation rate of filaments after 4 weeks in petridishesgenotype Sub 1 Sub 2 E192-1 1.0 4.7 D209-3 0.8 4.3 E207-2 4.8 6.5 E206-15.5 4.5 average 3.0 5.0

2.2.2 Double-Layer Cultivation

In a second experiment the white filaments or pieces thereof obtainedthrough cutting and comprising at least 2 axillary nodes are grown inglass jars containing 66 ml medium. The medium is MS0, which contains MSmicro+MS macro salts+MS vitamins, sucrose as the carbon source,myo-inositol, biotin, folic acid as additional, non MS vitamins andplant agar as a solidifying agent (for further details of the mediumcomposition see table A below).

About 5 filaments are placed in one jar, with the first node just belowthe surface of the agar. Then 2 ml liquid medium BI (MS micro+MS macrosalts+MS vitamins, sucrose as the carbon source, myo-inositol, biotin,folic acid as additional, non MS vitamins, and zeatin and BAP(6-benzylaminopurine) as growth regulators of the cytokinin family ofgrowth regulators (for further details of the medium composition seetable A below)) is added on top of the solid medium. For 100 ml of solidmedium 3 ml liquid B1 will be used. The jars are placed in the dark at18-22° C. for five weeks

At each subculture the proliferation rate or multiplication rate can bedetermined by dividing the number of filaments formed (after separatingor cutting the tissue back to the original number of nodes) by theinitial number of filaments.

2.3 Rooting

For rooting smaller explants are used. Each filament contains one or twoaxillary nodes. The explants are transferred to ventilated containers(ecoline boxes with XXL filter). The rooting medium is MS0-2 (halfstrength MS macro salts, full strength MS micro salts and MS vitamins,sucrose as the carbon source, myo-inositol, biotin, folic acid asadditional non-MS vitamins, and plant agar as the solidifying agents) orRAI2-2 (half strength MS macro salts, full strength MS micro salts andMS vitamins, sucrose as the carbon source, myo-inositol, biotin, folicacid as additional non-MS vitamins, indole-acetic acid, indole-butyricacid as growth regulators of the auxin family of growth regulators, andplant agar as the solidifying agents).

Both media can be used and will give similar results. Plants are rootedfor four weeks at a temperature of between 18° C. and 22° C. in a 16 hphotoperiod of app. 3000 lux. Rooting percentages are determined after4-6 weeks.

The rooted plantlets are taken out of the containers and washed in waterto remove traces of medium and agar. The plantlets are transferred tosoil in the greenhouse at 20° C. where they acclimatized and are treatedas normal tissue culture plants.

Example 3 Pelargonium 3.1 Plant Material and Etiolation

Pelargonium certified motherstock plants are grown in the greenhouse forat least 6 weeks under controlled phytosanitary conditions. Branches areobtained from said motherstock plants and used as explants foretiolation. Before sterilization all the leaves are removed. Theexplants are surface sterilized with a 1.3% sodium hypochlorite solution(30% commercial bleach) for 15 minutes and washed three times withsterile distilled water.

The defoliated and sterilized branches are cut into pieces such thateach resulting piece contains one or two nodes. Explants are placedhorizontally on medium in a petridish, with a maximum of three explantsper petridish. Petridishes are sealed with nescofilm.

For shoot multiplication and etiolation two different MS-based media(MS1 and MS2Z) are used. Both media can be suitably used for the shootmultiplication and etiolation of the Pelargonium explants. Certain ofthe tested genotypes appear to have preferences for one or the othermedium, however, overall the results obtained are very similar for bothmedia.

The MS1 medium contains MS micro+MS macro salts+MS vitamins, sucrose asa carbon source, myo-inositol, biotin, folic acid as additional, nonMS-vitamins, PVP (polyvinyl pyrrolidone) as a further supplement, BAP(6-benzylaminopurine), and 2iP (2-isopentenyl-adenine) as growthregulators of the cytokinin family of growth regulators and plant agaras a solidifying agent. The pH is set at 5.8 before autoclaving (forfurther details of the medium composition see table A below).

The MS2Z medium contains MS micro+MS macro salts+MS vitamins, sucrose asa carbon source, myo-inositol, biotin, folic acid as additional, nonMS-vitamins, zeatine as a growth regulator of the cytokinin family ofgrowth regulators and plant agar as the solidifying agent. The pH is setat 5.8 before autoclaving (for further details of the medium compositionsee table A below).

Explants are grown in the dark at a temperature of between 22° C. and24° C. for 3 to 4 weeks. In general 60%-80% of the explants will form afilament.

After three to four weeks in the dark the axillary shoots are etiolatedinto white filaments. Each filament had one or more axillary nodes.

3.2 Proliferation

The white filaments obtained in Example 3.1 are transferred to fresh MS1or MS2Z medium in petridishes for maintenance. They are cut into piecescontaining one or two nodes. For preserving the stock the petridishescan be placed in the dark at 4° C. for at least 4 months.

For an efficient proliferation each filament or pieces thereof obtainedthrough cutting contain at least two or three, but preferably fouraxillary nodes at the start of the subculture. The filaments are grownin glass jars containing 66 ml medium. The medium is MS0, which containMS micro+MS macro salts+MS vitamins, sucrose as a carbon source,myo-inositol, biotin, folic acid as additional non-MS vitamins, andplant agar as the solidifying agent (for further details of the mediumcomposition see table A below).

About 15±5 filaments are placed in one jar, with the first node justbelow the surface of the agar. Then 2 ml liquid medium KE (MS micro+MSmacro salts+MS vitamins, sucrose as a carbon source, myo-inositol,biotin, folic acid as additional non-MS vitamins, zeatin and thidiazuronas growth regulators of the cytokinin family of growth regulators (forfurther details of the medium composition see table A below)) is addedon top of the solid medium. For 100 ml of solid medium 3 ml liquid KEwill be used. The jars are placed in the dark at 22-24° C. for fourweeks. After four weeks the same procedure, as described above, forproliferation can be repeated.

At each subculture the proliferation rate or multiplication rate can bedetermined by dividing the number of filaments formed (after separatingor cutting the tissue back to the original number of nodes) by theinitial number of filaments. The proliferation rates are determined for10 genotypes for 3 subcultures (see table 5 below).

TABLE 5 Proliferation rates of 10 genotypes subculture 1 2 3 Apache 4.75.0 3.7 Mirage 5.2 5.0 4.7 Charlotte 4.7 5.1 4.2 Helios 5.2 5.2 3.7Olavi 5.4 4.9 3.6 Verseau 4.3 4.0 4.4 P. Blanche 4.7 5.5 3.9 Super Rose5.0 4.4 3.9 Philiomel 1 4.8 4.8 4.5 Balcon Rose 4.5 5.0 3.6 average 4.94.9 4.0

3.3 Rooting

For rooting smaller explants are used. Each filament contains one or twoaxillary nodes. The explants are transferred to ventilated containers(ecoline boxes with XXL filter). The rooting medium is MS0-2 (halfstrength MS macro salts, full strength MS micro salts and MS vitamins,sucrose as the carbon source, myo-inositol, biotin, folic acid asadditional non-MS vitamins, and plant agar as the solidifying agents) orRAI2-2 (half strength MS macro salts, full strength MS micro salts andMS vitamins, sucrose as the carbon source, myo-inositol, biotin, folicacid as additional non-MS vitamins, indole-acetic acid, indole-butyricacid as growth regulators of the auxin family of growth regulators, andplant agar as the solidifying agents).

Both media can be used and will give similar results. Plants are rootedfor four weeks at a temperature of between 22° C. and 24° C. in a 16 hphotoperiod of app. 3000 lux. Rooting percentages are determined for 10genotypes, after 4 weeks (see table 6 below).

TABLE 6 Rooting % of 10 genotypes genotype MS0-2 RAI2-2 Apache 93 100Helios 91 91 Charlotte 97 97 Olavi 90 85 Mirage 95 nd S. Rose 87 100 P.Blanche 79 93 Philiomel 1 100 100 B. Rose 88 83 Verseau 82 100 average90 94

The rooted plantlets are taken out of the containers and washed in waterto remove traces of medium and agar. The plantlets are transferred tosoil in the greenhouse at 20° C. where they acclimatized and are treatedas normal tissue culture plants.

TABLE A Media Compound MS0 MS0-2 MS1 MS1-2 MS2Z SP1 B1 KE RAI2-2 MacroElements MS MS half MS MS half MS MS mod. MS MS MS half KNO₃ 1.9 g · l⁻¹0.95 g · l⁻¹ 1.9 g · l⁻¹ 0.95 g · l⁻¹ 1.9 g · l⁻¹ 1.8 g · l⁻¹ 1.9 g ·l⁻¹ 1.9 g · l⁻¹ 0.95 g · l⁻¹ NH₄NO₃ 1.65 g · l⁻¹ 0.825 g · l⁻¹ 1.65 g ·l⁻¹ 0.825 g · l⁻¹ 1.65 g · l⁻¹ 0.4 g · l⁻¹ 1.65 g · l⁻¹ 1.65 g · l⁻¹0.825 g · l⁻¹ Ca(NO₃)₂ × 4H₂O — — — — — 1.2 g · l⁻¹ — — — CaCl₂ × 2H₂O0.44 g · l⁻¹ 0.22 g · l⁻¹ 0.44 g · l⁻¹ 0.22 g · l⁻¹ 0.44 g · l⁻¹ 0.44 g· l⁻¹ 0.44 g · l⁻¹ 0.22 g · l⁻¹ MgSO₄ × 7H₂O 0.37 g · l⁻¹ 0.185 g · l⁻¹0.37 g · l⁻¹ 0.185 g · l⁻¹ 0.37 g · l⁻¹ 0.36 g · l⁻¹ 0.37 g · l⁻¹ 0.37 g· l⁻¹ 0.185 g · l⁻¹ KH₂PO₄ 0.17 g · l⁻¹ 0.085 g · l⁻¹ 0.17 g · l⁻¹ 0.085g · l⁻¹ 0.17 g · l⁻¹ 0.27 g · l⁻¹ 0.17 g · l⁻¹ 0.17 g · l⁻¹ 0.085 g ·l⁻¹ Micro-Elements MS MS MS MS MS MS MS MS MS vitamins MS MS MS MS MS MSMS MS MS sucrose 30 g · l⁻¹ 30 g · l⁻¹ 30 g · l⁻¹ 30 g · l⁻¹ 30 g · l⁻¹20 g · l⁻¹ 30 g · l⁻¹ 30 g · l⁻¹ 10 g · l⁻¹ myo-inositol 0.1 g · l⁻¹ 0.1g · l⁻¹ 0.1 g · l⁻¹ 0.1 g · l⁻¹ 0.1 g · l⁻¹ 0.1 g · l⁻¹ 0.1 g · l⁻¹ 0.1g · l⁻¹ 0.1 g · l⁻¹ biotin 0.05 mg · l⁻¹ 0.05 mg · l⁻¹ 0.05 mg · l⁻¹0.05 mg · l⁻¹ 0.05 mg · l⁻¹ 0.1 mg · l⁻¹ 0.05 mg · l⁻¹ 0.05 mg · l⁻¹0.05 mg · l⁻ folic acid 0.5 mg · l⁻¹ 0.5 mg · l⁻¹ 0.5 mg · l⁻¹ 0.5 mg ·l⁻¹ 0.5 mg · l⁻¹ 0.5 mg · l⁻¹ 0.5 mg · l⁻¹ 0.5 mg · l⁻¹ ¹0.5 mg · l⁻¹PVP (polyvinyl 0.5 g · l⁻¹ 0.5 g · l⁻¹ — pyrrolidone) BA (6- 0.1 mg ·l⁻¹ 0.1 mg · l⁻¹ 0.25 mg · l⁻¹ 5.0 mg · l⁻¹ benzylaminopurine) 2iP(2-isopentenyl- 0.1 mg · l⁻¹ 0.1 mg · l⁻¹ adenine) zeatine 0.2 mg · l⁻¹0.2 mg · l⁻¹ 0.2 mg · l⁻¹ thidiazuron (TDZ) 0.05 mg · l⁻¹ IAA (Indole-3-0.1 mg · l⁻¹ 0.5 mg · l⁻¹ Acetic Acid) IBA (Indole-Butyric 0.5 mg · l⁻¹Acid) plant agar 6 g · l⁻¹ 6 g · l⁻¹ 6 g · l⁻¹ 6 g · l⁻¹ 6 g · l⁻¹ 6 g ·l⁻¹ 6 g · l⁻¹ pH 5.8 (before 5.8 (before 5.8 (before 5.8 (before 5.8(before 5.8 (before 5.8 (before 5.8 (before 5.8 (before autoclav)autoclav) autoclav) autoclav) autoclav) autoclav) autoclav) autoclav)autoclav)

1. Method for the production of young plants and/or of micro-parentstock of plants belonging to the group of herbaceous ornamentals, whichmethod comprises an in vitro culture phase during which explantsobtained from parent stock of species to be propagated, or derivativesof these explants, are subjected to micropropagation which is carriedout under suitable conditions and on suitable culture media, in order toproduce micro-plantlets which, when subjected to an in vivo culturephase, are intended to develop into plants or into micro-parent stock,characterized in that, to carry out said micropropagation: a) explantsare obtained from parent stock of species to be propagated, or fromderivatives of these explants, b) the explants are, under axenicconditions, in the dark and for a suitable period of time, placed on ashoot initiation medium which is composed such as to suit each plantspecies to be propagated, resulting in the induction of white a firstgeneration of filament formation which first generation of filamentscomprise one or more axillary buds, c) optionally, each of the whitefilaments is divided into a plurality of pieces, each of which comprisesan axillary bud, but at least one axillary bud, and said filamentsand/or pieces obtained from a first generation of white filaments arereturned under axenic conditions and in the dark to a proliferationmedium for a suitable period of time sufficient to allow the tissue ofthe plant cuttings to multiply, preferably exponentially, and to producea large quantity of etiolated white filaments comprising a high numberof axillary nodes, so as to induce the formation of a new generation ofwhite filaments, and the procedure is repeated as often as necessary toproduce different consecutive generations of white filaments until thedesired amount of white filaments has been obtained, e) and saidfilaments and/or pieces are, in the light, under axenic conditions andfor a suitable period of time, placed on or into a rooting medium, whichallows each axillary bud to produce a micro-plantlet which has roots. 2.Method according to claim 1, characterized in that meristems taken fromparent stock are used as explants which are grown under axenicconditions in the light for a first period of time on a developmentmedium common to each plant variety to be propagated, in order to bringabout the formation of little plantlets which have first leaf primordiaand then, for a second period of time, on a rooting medium common toeach plant variety, in order to obtain rooted plantlets which are freefrom bacteria and viruses and which are intended to be placed on theproliferation medium in the dark in order to produce said whitefilaments.
 3. Method according to claim 1, characterized in thatmicro-cuttings taken from certified, bacteria- and virus-free parentstock are used as explants.
 4. Method according to claim 1,characterized in that the filaments and/or pieces thereof obtained froma first generation of white filaments are returned under axenicconditions to a fresh proliferation medium and grown in the dark for asuitable period of time so as to induce the formation of a newgeneration of white filaments, and that the procedure is repeated asoften as necessary to produce different consecutive generations of whitefilaments until the desired amount of white filaments has been obtained.5. Method according to claim 1, characterized in that a high propagationrate is maintained over one or more, but specifically over up to 10propagation cycles in the dark.
 6. Method according to claim 1,characterized in that a two-layer cultivation system is employed inorder to promote shoot multiplication characterized in that the shootinitiation and/or the proliferation medium is provided in form of a twolayer system comprising a solidified bottom layer comprising essentiallymacro- and micro-elements, vitamins and a carbon source, but no growthregulator, which bottom layer is overlayed with a fluid top layercomprising a growth regulator, which supports shoot multiplication bypromoting the breakage and development of axillary buds into etiolatedshoots and further into white filaments and/or the growth, butespecially the elongation growth of the developing shoots into shootscomprising a high number of axillary nodes.
 7. Method according to claim1, characterized in that the number of axillary buds of a while filamentdeveloping from an initial node is at least between 2 and 10 axillarybuds/per initial node.
 8. Method according to claim 1, characterized inthat a culture medium of the “MS” type is used as the shoot initiationand/or proliferation medium, supplemented with a growth regulator beingprovided in a concentration which supports shoot multiplication bypromoting the breakage and development of axillary buds into etiolatedshoots and further into white filaments and/or the growth, butespecially the elongation growth of the developing shoots into shootscomprising a high number of axillary nodes.
 9. Method according to claim1, characterized in that the shoot initiation and/or proliferationmedium comprises a growth regulator belonging to the cytokinin family ofgrowth regulators or a combination of growth regulators belonging to thecytokinin family of growth regulators and to the auxin family of growthregulators.
 10. Method according to claim 9, characterized in that thegrowth regulators belonging to the cytokinin family of growth regulatorsor to the auxin family of growth regulators are provided in aconcentration of between 0.01 mg/l and 5.0 mg/l.
 11. Method according toclaim 1, characterized in that the white filaments are, at ambienttemperature, and in the dark, stored in a proliferation medium which isperiodically changed, before being subjected to the rooting phase,during which each bud produces a rooted micro-plantlet intended toprovide a young plant or a micro-parent stock plant.
 12. Methodaccording to claim 11, characterized in that the white filaments arestored at a temperature of between 2° C. and 10° C. for at least up to10 month before being subjected to the rooting phase.
 13. Methodaccording to claim 1, characterized in that plants selected from thegroup consisting of pelargonium, petunia including cascading petunia,poinsettia, cyclamen, chrysanthemum, busy Lizzie, verbena, and torenia,are employed in the method according to the preceding claims.
 14. Youngplant which is distinguished in particular by an increased branchingrate, rapid growth, compact foliage, auto-regulation of the plant,characterized in that it is obtained from a micro-plantlet obtained froman explant, or derivative of an explant of the plant to be propagated,subjected to the culture conditions of the method according to claim 1.15. Young plant according to claim 14, characterized in that said plantis selected from the group consisting of pelargonium, petunia includingcascading petunia, poinsettia, cyclamen, chrysanthemum, busy Lizzie,verbena, and torenia.
 16. Method according to claim 2, characterized inthat the filaments and/or pieces thereof obtained from a firstgeneration of white filaments are returned under axenic conditions to afresh proliferation medium and grown in the dark for a suitable periodof time so as to induce the formation of a new generation of whitefilaments, and that the procedure is repeated as often as necessary toproduce different consecutive generations of white filaments until thedesired amount of white filaments has been obtained.